EP2, a receptor for PGE2, regulates tumor angiogenesis through direct effects on endothelial cell motility and survival

Prostaglandin E2 (PGE2), a major cyclooxygenase (COX) metabolite, plays important roles in tumor biology. We studied the role of EP2, a receptor for PGE2, in tumor angiogenesis using EP2 knockout mice. We found that deletion of the EP2 receptor impaired tumor angiogenesis and this finding was confirmed by an in vivo corneal angiogenesis model and an ex vivo aortic ring assay. To further characterize the cellular mechanisms of the EP2 receptor in angiogenesis, we isolated primary pulmonary endothelial cells (ECs) from wild-type (wt) and EP2-/- mice and observed that EP2-/- ECs exhibited defects in vascular branch formation when compared to wt ECs. In addition, EP2-/- ECs showed impaired cell motility on collagen-coated surface and they responded poorly to PGE2-induced cell migration compared to control cells. However, no difference in cell proliferation was observed between the EP2-/- and wt Ecs. In addition, EP2-/- ECs were more susceptible to apoptosis than wt cells under growth factor depletion conditions. Collectively, our data demonstrate that EP2 signaling in endothelium directly regulates tumor angiogenesis by contributing to cell survival and endothelial cell motility. Moreover, our finding suggests that EP2 is a major receptor in PGE2-mediated cell motility in ECs.     

环氧化酶2抑制剂对宫颈癌 Hela 细胞中 HPV18- E6和 COX -2表达的影响

目的：探讨环氧化酶2抑制剂 Celecoxib 对宫颈癌细胞 Hela 中 HPV18- E6、COX -2表达及其细胞增殖和凋亡的影响。方法：用不同浓度 Celecoxib 处理 Hela 细胞株后,免疫细胞化学 SP 法检测 HPV18- E6和COX -2蛋白的表达变化。RT - PCR 检测 HPV18- E6及 COX -2 mRNA 表达的改变。MTT 法检测其对细胞增殖的影响。流式细胞仪测定细胞凋亡情况。Hoechst33258染色观察细胞的形态学变化。结果：Celecoxib 作用 Hela 细胞后 HPV18- E6和 COX -2基因 mRNA 及蛋白表达量明显低于对照组,且随着药物浓度增加表达呈梯度下降。各组之间差异具有显著性（P <0.05）,且两者之间具有显著相关性（P <0.01）。Celecoxib 抑制Hela 细胞增殖,作用呈量-效关系（P <0.05）。流式细胞仪显示各实验组凋亡率随浓度升高而增加,与对照组之间有显著差异（P <0.05）。Hoechst33258荧光染色显示经20μmol/ L 药物作用 Hela 细胞24h 后表现为典型调亡小体。结论：COX -2抑制剂 Celecoxib 可以同时下调宫颈癌 Hela 细胞中 HPV18- E6和 COX -2表达、抑制 Hela 细胞的增殖、促进其凋亡。     

下调FAM111B对乳腺癌细胞增殖和凋亡的影响

目的:探讨下调FAM111B对乳腺癌细胞系MDA-MB-231和MCF7细胞增殖和凋亡的影响及其机制。方法:构建siR-FAM111B慢病毒载体,转染乳腺癌MDA-MB-231和MCF7细胞,qRT-PCR检查转染组与对照组FAM111B mRNA表达,Western blotting法检测各组细胞FAM111B蛋白表达。用CCK-8法检测细胞的增殖能力。流式细胞术检测Annexin-V/PI双染各组细胞的凋亡情况。Western blotting法检测凋亡相关蛋白Bax和Bcl-2的表达。结果:siR-FAM111B成功转染MDA-MB-231和MCF7细胞,转染后应用qRT-PCR和Western blotting检测,结果显示,FAM111B mRNA与蛋白水平均下调。siR-FAM111B能抑制两种细胞的增殖。Annexin-V/PI双染结果显示,下调FAM111B诱导两种细胞凋亡。Western blotting结果显示,下调FAM111B可以促进两种细胞Bax的表达,抑制Bcl-2的表达。结论:下调FAM111B能够抑制乳腺癌细胞的增殖,通过调节线粒体凋亡通路诱导细胞凋亡。     

Extracellular signal-regulated kinases and AP-1 mediate the up-regulation of vascular endothelial growth factor by PDGF in human vascular smooth muscle cells

Endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) have been shown to communicate with each other via cytokine signaling during neovascularization. In this study, we investigated the effect of platelet-derived growth factor (PDGF), a cytokine released from tumors and ECs, on vascular endothelial growth factor (VEGF) expression in human VSMCs and underlying signal transduction pathways. PDGF induced VEGF expression in a time- and concentration-dependent manner. PDGF induced the activation of extra-cellular signal-regulated kinase-1/2 (ERK-1/2), but not the activation of c-jun amino terminal kinase (JNK) and P38 mitogen-activated protein kinase (MAPK). Specific inhibitor of mitogen-activated protein kinase kinase (MEK)-1 was found to suppress VEGF expression and promoter activity. The expression of vectors encoding a mutated-type MEK-1 decreased the VEGF promoter activity. Electrophoretic mobility shift assay revealed that PDGF dose-dependently increased the DNA binding activity of AP-1. Transient transfection studies using an AP-1 decoy oligonucleotide confirmed that the activation of AP-1 is involved in PDGF-induced VEGF upregulation. Conditioned media from the human VSMCs pretreated with PDGF could remarkably stimulate the in vitro growth of human umbilical vein endothelial cells and this effect was partially abrogated by VEGF neutralizing antibodies. The above results suggest that ERK-1/2 and AP-1 signaling pathways are involved in the PDGF-induced VEGF expression in human VSMCs and that these paracrine signaling pathways induce endothelial cell proliferation.     

Selective inhibition of cyclooxygenase (COX)-2 inhibits endothelial cell proliferation by induction of cell cycle arrest

High-level expression of cyclooxygenase (COX)-2 is reported in 80-90% of colorectal adenocarcinomas. Selective inhibition of COX-2 was shown to reduce colorectal tumorigenesis in different models of carcinogenesis and to prevent metastasis in xenograft tumor models, as well as to suppress in vitro induced angiogenesis. Recently, COX-2 was reported to be expressed not only in malignant epithelial cells, but also in the neovasculature that feeds the tumor in a variety of solid human cancers. Thus, one of the possible mechanisms by which selective COX-2 inhibitor reduces tumor growth and metastasis is through inhibition of tumor angiogenesis. Although a report suggested a possible role of endothelial COX-1 in the process of angiogenesis, in a recent study, the selective inhibition of COX-2 was shown to strongly inhibit angiogenesis by inducing endothelial cell (EC) apoptosis. In the present study, using human umbilical vein endothelial cells (HUVECs) as a model of angiogenesis, we investigated the potential antiangiogenic effect of the selective COX-2 inhibitor and its mechanism of action, and clearly demonstrated that selective inhibition of COX-2 caused a dose-dependent decrease in the proliferative activity of ECs, as well as an inhibition of capillary-like tube formation. The inhibitory effect on EC proliferation was dependent on the cell cycle arrest to the G1 phase and not on cell apoptosis.     

红景天苷通过miRNA-210-3p/E2F3抑制非小细胞肺癌细胞的增殖和迁移

目的:探讨红景天苷（salidroside，Sal）通过miRNA-210-3p/E2F3对非小细胞肺癌（non-small cell lung cancer，NSCLC）细胞增殖和迁移的影响及作用机制。方法:通过生物信息网站分析miR-210-3p及其靶基因在肺腺癌组织和正常组织中的表达及生存影响。采用免疫组化法检测肺腺癌组织和正常组织中E2F3蛋白的表达；qRT-PCR检测NSCLC细胞中miR-210-3p和E2F3基因表达；Western blot检测NSCLC细胞中E2F3蛋白表达；CCK-8、细胞划痕及克隆形成实验分别检测红景天苷和miR-210-3p对NSCLC细胞增殖、迁移及克隆形成的影响。结果:生物信息网站显示miR-210-3p和E2F3在肺腺癌组织中高表达，且与不良预后相关。Sal以时间-浓度依赖抑制NSCLC细胞的增殖活性（P＜0.05），并显著降低NSCLC细胞的迁移及克隆形成能力（P＜0.05），而低浓度Sal对正常肺上皮细胞的增殖无明显抑制效果。miRNA-210-3p和E2F3表达在NSCLC细胞中上调（P＜0.05），Sal可抑制二者表达（P＜0.05）。与对照组比较，miR-210-3p inhibitor组E2F3表达上调（P＜0.05），miR-210-3p mimics组E2F3表达被抑制（P＜0.05），Sal与miR-210-3p mimics联用进一步抑制了miR-210-3p mimics所致的E2F3表达下调和细胞增殖和迁移的增强作用（P＜0.05）。结论:miR-210-3p和E2F3在肺腺癌中高表达，Sal可通过时间-浓度依赖负调控miRNA-210-3p/E2F3影响 NSCLC细胞的生物学行为，发挥抗癌作用。     

Radiosensitization of human vascular endothelial cells through Hsp90 inhibition with 17-N-allilamino-17-demethoxygeldanamycin

PURPOSE: In addition to invasive tumor cells, endothelial cells (ECs) of the tumor vasculature are an important target for anticancer radiotherapy. The purpose of the present work is to investigate how 17-N-allilamino-17-demethoxygeldanamycin (17AAG), known as an anticancer drug inhibiting heat shock protein 90 (Hsp90), modifies radiation responses of human vascular ECs. METHODS AND MATERIALS: The ECs cultured from human umbilical veins were exposed to gamma-irradiation, whereas some EC samples were pretreated with growth factors and/or 17AAG. Postirradiation cell death/survival and morphogenesis were assessed by means of terminal deoxynucleotidyl transferase biotin-deoxyuridine triphosphate nick end labeling or annexin V staining and clonogenic and tube-formation assays. The 17AAG-affected expression and phosphorylation of radioresistance-related proteins were probed by means of immunoblotting. Dominant negative or constitutively activated Akt was transiently expressed in ECs to manipulate Akt activity. RESULTS: It was found that nanomolar concentrations of 17AAG sensitize ECs to relatively low doses (2-6 Gy) of gamma-irradiation and abolish the radioprotective effects of vascular endothelial growth factor and basic fibroblast growth factor. The drug-induced radiosensitization of ECs seems to be caused by prevention of Hsp90-dependent phosphorylation (activation) of Akt that results in blocking the radioprotective phosphatidylinositol 3-kinase/Akt pathway. CONCLUSIONS: Clinically achievable concentrations of 17AAG can decrease the radioresistance intrinsic to vascular ECs and minimize the radioprotection conferred upon them by tumor-derived growth factors. These findings characterize 17AAG as a promising radiosensitizer for the tumor vasculature.     

Bovine papillomavirus type-2 DNA and expression of E5 and E7 oncoproteins in vascular tumours of the urinary bladder in cattle

Cattles suffering from chronic enzootic haematuria frequently develop urinary bladder tumours of both epithelial and mesenchymal origin mainly haemangioma and its malignant counterpart. The role of the bovine papillomavirus type-2 (BPV-2) and of its major transforming oncoprotein in naturally occurring urothelial carcinogenesis has been recently clarified. E5 interacts in vivo as in vitro with the beta receptor for the platelet-derived growth factor (PDGF). However, studies regarding tumours of mesenchymal origin such as those arising from blood vessels are lacking. We show that the BPV-2 is present in 100% of the vascular tumours of the urinary bladder examined. Twenty-six out of twenty-seven tumour samples (96%) expressed E5 while 20 out of 27 (74%) tumour samples expressed E7. The two viral oncoproteins were not expressed in normal endothelial cells. Additionally, they co-localize in neoplastic endothelial cells as demonstrated by confocal immunofluorescence. PDGFbeta receptor was also shown to be expressed and co-localizes with E5 in neoplastic blood vessels. Our results demonstrate, for the first time, that the BPV-2 is present in high percentage in tumours of mesenchymal origin arising in its natural host. Furthermore, the expression of the two viral oncoproteins confirm that the virus may have a causative role in the neoplastic process.     

E2F3a在U251胶质瘤细胞中的功能研究

背景与目的：E2F3a是一种重要的转录激活因子，在多种肿瘤组织中均呈现表达上调。E2F3a在恶性化程度较高的神经胶质瘤中表达较高，目前它在胶质瘤细胞中的功能尚不清楚。本文的目的是研究E2F3。对U251胶质瘤细胞周期和凋亡的影响。方法：通过脂质体介导质粒转染在U251细胞中过表达E2F3a：用Western blotting检测细胞内E2F3a蛋白质的表达水平：用PT-核DNA染色结合流式细胞术分析细胞周期分布：用Annexin V—PE和7-AAD染色结合流式细胞术分析细胞凋亡比例；用实时荧光定量PCR方法测定细胞内A2、B1、D1和E细胞周期素mRNA的相对表达水平。结果：与空载体转染对照组相比．E2F3a过表达可将位于S期的细胞比例提高28％（P〈0．01），将位于G0/G1期和G2/M的细胞比例分别降低15％和13％（P〈0．01）。E2F3a过表达对细胞凋亡无明显影响。进一步研究发现，E2F3a可显著上调细胞周期素D1和E的mRNA表达．而不影响周期素A2和B1的mRNA表达。结论：E2F3a是胶质瘤细胞增殖的促进因子，它可通过上调细胞周期素D1和E的表达加速细胞由G，期进入S期。     

E‐cadherin expression increases cell proliferation by regulating energy metabolism through nuclear factor‐κB in AGS cells

β‐Catenin is a central player in Wnt signaling, and activation of Wnt signaling is associated with cancer development. E‐cadherin in complex with β‐catenin mediates cell–cell adhesion, which suppresses β‐catenin‐dependent Wnt signaling. Recently, a tumor‐suppressive role for E‐cadherin has been reconsidered, as re‐expression of E‐cadherin was reported to enhance the metastatic potential of malignant tumors. To explore the role of E‐cadherin, we established an E‐cadherin‐expressing cell line, EC96, from AGS cells that featured undetectable E‐cadherin expression and a high level of Wnt signaling. In EC96 cells, E‐cadherin re‐expression enhanced cell proliferation, although Wnt signaling activity was reduced. Subsequent analysis revealed that nuclear factor‐κB (NF‐κB) activation and consequent c‐myc expression might be involved in E‐cadherin expression‐mediated cell proliferation. To facilitate rapid proliferation, EC96 cells enhance glucose uptake and produce ATP using both mitochondria oxidative phosphorylation and glycolysis, whereas AGS cells use these mechanisms less efficiently. These events appeared to be mediated by NF‐κB activation. Therefore, E‐cadherin re‐expression and subsequent induction of NF‐κB signaling likely enhance energy production and cell proliferation.